Based on evidence from previous studies that activation of NOD2 by muramyl dipeptide (MDP) leads to the down-regulation of TLR2 responses and that the absence of such regulation leads to heightened Th1 responses, in initial studies we treated mice with either dextran-sulfate-induced colitis (DSS-colitis) or trinitrobenzene sulfonic acid-induced colitis (TNBS-colitis) with MDP to determine the latters effect on inflammation in vivo. Indeed, we found that MDP administration greatly decreased the inflammation in both of these models.[unreadable] [unreadable] The basis of this down-regulatory effect then became apparent in subsequent in vitro studies of dendritic cells in which we showed that pre-stimulation of the cells with MDP rendered the cells unresponsive to a wide variety of TLR stimulants in assays of a spectrum of inflammatory cytokines. Subsequent studies designed to reveal the mechanism of this effect showed that MDP stimulation of NOD2 induced the expression of IRF-4, a factor previously shown to inhibit TLR responses and that gene silencing of IRF-4 led to restoration of responses.[unreadable] [unreadable] With this insight in hand we then returned to in vivo studies of experimental colitis and showed that in vivo down-regulation of IRF-4 by administration of IRF-4-specific siRNA encapsulated in a viral envelope to facilitate cell entry abolished the effect of MDP on TNBS-colitis. In addition, it abolished the down-regulatory effect of MDP administration on TLR ligand cytokine induction. In addition, we showed that MDP administration was unable to affect DSS-colitis induced in IRF-4-deficient mice. [unreadable] [unreadable] In a final series of studies relating these findings to Crohns disease we re-constituted NOD2-deficient mice with plasmids expressing either wild type NOD2 or NOD2 bearing a frameshift mutation identical to that found in Crohns disease. We then showed that MDP administration prevented DSS-colitis in mice reconstituted with wild type NOD2 but not mice reconstituted with mutated (frameshift mutation) NOD2. Thus, the frameshift mutation conferred a phenotype characterized by an inability to acquire MDP-induced unresponsiveness.